GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles.

GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca2+-dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the "Flash and Freeze-fracture" method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals.

The crustacean DNA virus tegument protein VP26 binds to SNAP29 to inhibit SNARE complex assembly and autophagic degradation.

Autophagy generally functions as a cellular surveillance mechanism to combat invading viruses, but viruses have evolved various strategies to block autophagic degradation and even subvert it to promote viral propagation. White spot syndrome virus (WSSV) is the most highly pathogenic crustacean virus, but little is currently known about whether crustacean viruses such as WSSV can subvert autophagic degradation for escape. Here, we show that even though WSSV proliferation triggers the accumulation of autophagosomes, autophagic degradation is blocked in the crustacean species red claw crayfish. Interestingly, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex including CqSNAP29, CqVAMP7, and the novel autophagosome SNARE protein CqSyx12 is required for autophagic flux to restrict WSSV replication, as revealed by gene silencing experiments. Simultaneously, the expressed WSSV tegument protein VP26, which likely localizes on autophagic membrane mediated by its transmembrane region, binds the Qb-SNARE domain of CqSNAP29 to competitively inhibit the binding of CqSyx12-Qa-SNARE with CqSNAP29-Qb-SNARE; this in turn disrupts the assembly of the CqSyx12-SNAP29-VAMP7 SNARE complex, which is indispensable for the proposed fusion of autophagosomes and lysosomes. Consequently, the autophagic degradation of WSSV is likely suppressed by the expressed VP26 protein in vivo in crayfish, thus probably protecting WSSV components from degradation via the autophagosome-lysosome pathway, resulting in evasion by WSSV. Collectively, these findings highlight how a DNA virus can subvert autophagic degradation by impairing the assembly of the SNARE complex to achieve evasion, paving the way for understanding host-DNA virus interactions from an evolutionary point of view, from crustaceans to mammals.IMPORTANCEWhite spot syndrome virus (WSSV) is one of the largest animal DNA viruses in terms of its genome size and has caused huge economic losses in the farming of crustaceans such as shrimp and crayfish. Detailed knowledge of WSSV-host interactions is still lacking, particularly regarding viral escape from host immune clearance. Intriguingly, we found that the presence of WSSV-VP26 might inhibit the autophagic degradation of WSSV in vivo in the crustacean species red claw crayfish. Importantly, this study is the first to show that viral protein VP26 functions as a core factor to benefit WSSV escape by disrupting the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which is necessary for the proposed fusion of autophagosomes with lysosomes for subsequent degradation. These findings highlight a novel mechanism of DNA virus evasion by blocking SNARE complex assembly and identify viral VP26 as a key candidate for anti-WSSV targeting.

PcEiger links the Imd/Relish pathway to ROS production in the intestine of the red swamp crayfish.

In the arthropod gut, commensal microbiota maintain the immune deficiency (Imd)/Relish pathway for expression of antimicrobial peptides, whereas pathogenic bacteria induce dual oxidase 2 (Duox2) for production of extracellular microbicidal reactive oxygen species (ROS). The Imd/Relish pathway and the Duox2/ROS system are regarded as independent systems. Here, we report that these two systems are bridged by the tumor necrosis factor (TNF) ortholog PcEiger in the red swamp crayfish Procambarus clarkii. PcEiger expression is induced by commensal bacteria or the Imd/Relish pathway. PcEiger knockdown alters bacterial abundance and community composition due to variations in the oxidative status of the intestine. PcEiger induces Duox2 expression and ROS production by regulating the activity of the transcription factor Atf2. Moreover, PcEiger mediates regulation of the Duox2/ROS system by commensal bacteria and the Imd/Relish pathway. Our findings suggest that the Imd/Relish pathway regulates the Duox2/ROS system via PcEiger in P. clarkii, and they provide insights into the crosstalk between these two important mechanisms for arthropod intestinal immunity.

Effects of hydroxy methionine zinc on growth performance, immune response, antioxidant capacity, and intestinal microbiota of red claw crayfish (Procambarus clarkii).

This study aimed to evaluate the effects of varying zinc (Zn) levels on the growth performance, non-specific immune response, antioxidant capacity, and intestinal microbiota of red claw crayfish (Procambarus clarkii (P. clarkii)). Adopting hydroxy methionine zinc (Zn-MHA) as the Zn source, 180 healthy crayfish with an initial body mass of 6.50 ± 0.05 g were randomly divided into the following five groups: X1 (control group) and groups X2, X3, X4, and X5, which were fed the basal feed supplemented with Zn-MHA with 0, 15, 30, 60, and 90 mg kg-1, respectively. The results indicated that following the addition of various concentrations of Zn-MHA to the diet, the following was observed: Specific growth rate (SGR), weight gain rate (WGR), total protein (TP), total cholesterol (TC), the activities of alkaline phosphatase (AKP), phenoloxidase (PO), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD) and catalase (CAT), the expression of CTL, GPX, and CuZn-SOD genes demonstrated a trend of rising and then declining-with a maximum value in group X4-which was significantly higher than that in group X1 (P < 0.05). Zn deposition in the intestine and hepatopancreas, the activity of GSH-PX, and the expression of GSH-PX were increased, exhibiting the highest value in group X5. The malonaldehyde (MDA) content was significantly reduced, with the lowest value in group X4, and the MDA content of the Zn-MHA addition groups were significantly lower than the control group (P < 0.05). In the analysis of the intestinal microbiota of P. clarkii, the number of operational taxonomic units in group X4 was the highest, and the richness and diversity indexes of groups X3 and X4 were significantly higher than those in group X1 (P < 0.05). Meanwhile, the dietary addition of Zn-MHA decreased and increased the relative abundance of Proteobacteria and Tenericutes, respectively. These findings indicate that supplementation of dietary Zn-MHA at an optimum dose of 60 mg kg-1 may effectively improve growth performance, immune response, antioxidant capacity, and intestinal microbiota richness and species diversity in crayfish.

Evidence of interregional similarity in crayfish metabolomes at reference sites: Progress towards the metabolome as a biomonitoring tool.

It has been proposed that biomonitoring may benefit from the use of metabolomics (the study of all small molecules in an organism) to detect sub-lethal organism stress through changes in the metabolite profile (i.e., the metabolome). However, to integrate the metabolome into biomonitoring programs the amount of natural variability among and within populations of indicator taxa must be established prior to generating a reference condition. This study determined variation in the metabolome among ecoregion and stream of origin in the northern crayfish (Faxonius virilis) and if that variation inhibited detection of stressor effects at sites exposed to human activities. We collected crayfish from seven minimally disturbed streams (i.e., reference streams), distributed across three level II ecoregions in central Canada and compared their metabolomes. We found ecoregion and stream origin were poor predictors of crayfish metabolomes. This result suggests crayfish metabolomes were similar, despite differing environmental conditions. Metabolomes of crayfish collected from three stream sites exposed to agricultural activity and municipal wastewater (i.e., test sites) were then compared to the crayfish metabolomes from the seven reference streams. Findings showed that crayfish metabolomes from test sites were strongly differentiated from those at all reference sites. The consistency in the northern crayfish metabolome at the studied reference streams indicates that a single reference condition may effectively detect impacts of human activities across the sampled ecoregions.

De Novo Cloning and Functional Characterization of a Mechanosensitive Piezo-Like Ion Channel in the Crayfish.

BACKGROUND/AIMS: Mechanosensitive ion channels are the principal elements in the transduction of mechanical force to neural activity. To date, considerably fewer studies have been published about the molecular and structural properties of mechanosensitive channels. Piezo channels are the only ion channel family in eukaryotes which is selectively gated by the membrane tension. Piezo channels have been described in mammals and some other eukaryotes. However, not much information is available for the crustaceans. METHODS: Conventional cloning methods were used to clone the putative PIEZO channel mRNA in crayfish ganglia samples. HEK293T cells were transfected by the plasmid of the cloned gene for functional studies. The CDS of the mRNA translated into the protein sequence and three-dimensional structure of the channel has been calculated. RESULTS: An mRNA, 9378 bp, was firstly cloned from crayfish which codes a 2674 residues protein. The cloned sequence is similar to the piezo channel mRNAs reported in the other species. The sequence of the coded protein has been analyzed, and some functional domains have been identified. A three-dimensional structure of the coded protein was successfully calculated in reference to mouse piezo 1 channel protein data. A plasmid with a fluorescent protein indicator was synthesized for heterologous expression in HEK293T cells. The evoked calcium response to mechanical stimulation was not different from those observed in the control cells. However, the transfected cells were more sensitive to the gating modifier YODA-1. CONCLUSION: Based on the apparent similarity in sequence, structure and functional properties to other known piezo channels, it has been proposed that cloned mRNA may code a piezo-like ion channel in crayfish.

Comparative transcriptome analysis of differentially expressed genes and pathways in Procambarus clarkii (Louisiana crawfish) at different acute temperature stress.

Procambarus clarkii is an important economic species in China, and exhibit heat and cold tolerance in the main culture regions. To understand the mechanisms, we analyzed the hepatopancreas transcriptome of P. clarkii treated at 10 °C, 25 °C, and 30 °C, then 2092 DEGs and 6929 DEGs were found in 30 °C stress group and 10 °C stress group, respectively. KEGG pathway enrichment results showed that immune pathway is the main stress pathway for 10 °C treatment and metabolic pathway is the main response pathway for 30 °C treatment, which implies low temperature stress induces the damage of the immune system and increases the susceptibility of bacteria while the body response to high temperature stress through metabolic adjustment. In addition, flow cytometry proved that both high and low temperature stress caused different degrees of apoptosis of hemocytes, and dynamic transcription heat map analysis also identified the differential expression of HSPs family genes and apoptosis pathway genes under different heat stresses. This indicates that preventing damaged protein misfolding and accelerating cell apoptosis are necessary mechanisms for P. clarkii to cope with high and low temperature stress. Our research has deepened our understanding of the complex molecular mechanisms of P. clarkii in response to acute temperature stress, and provided a potential strategy for aquatic animals to relieve environmental duress.

Transcriptomic Changes Following Induced De-Masculinisation of Australian Red Claw Crayfish Cherax quadricarinatus.

The Australian red claw crayfish Cherax quadricarinatus, an emerging species within the freshwater aquaculture trade, is not only an ideal species for commercial production due to its high fecundity, fast growth, and physiological robustness but also notoriously invasive. Investigating the reproductive axis of this species has been of great interest to farmers, geneticists, and conservationists alike for many decades; however, aside from the characterisation of the key masculinising insulin-like androgenic gland hormone (IAG) produced by the male-specific androgenic gland (AG), little remains known about this system and the downstream signalling cascade involved. This investigation used RNA interference to silence IAG in adult intersex C. quadricarinatus (Cq-IAG), known to be functionally male but genotypically female, successfully inducing sexual redifferentiation in all individuals. To investigate the downstream effects of Cq-IAG knockdown, a comprehensive transcriptomic library was constructed, comprised of three tissues within the male reproductive axis. Several factors known to be involved in the IAG signal transduction pathway, including a receptor, binding factor, and additional insulin-like peptide, were found to not be differentially expressed in response to Cq-IAG silencing, suggesting that the phenotypic changes observed may have occurred through post-transcriptional modifications. Many downstream factors displayed differential expression on a transcriptomic level, most notably related to stress, cell repair, apoptosis, and cell proliferation. These results suggest that IAG is required for sperm maturation, with necrosis of arrested tissue occurring in its absence. These results and the construction of a transcriptomic library for this species will inform future research involving reproductive pathways as well as biotechnological developments in this commercially and ecologically significant species.

Transcriptome Analysis Reveals the Effect of Stocking Density on Energy Metabolism in the Gills of Cherax quadricarinatus under Rice-Crayfish Co-Culture.

Stocking density is a crucial factor affecting productivity in aquaculture, and high stocking density is a stressor for aquatic animals. In this study, we aimed to investigate the effects of stocking densities on oxidative stress and energy metabolism in the gills of Cherax quadricarinatus under rice-crayfish farming. The C. quadricarinatus were reared at low density (LD), medium density (MD), and high density (HD) for 90 days. The results showed that the superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA) levels were higher in the HD group than those in the LD group. Transcriptomic analysis revealed 1944 upregulated and 1157 downregulated genes in the gills of the HD group compared to the LD group. Gene ontology (GO) enrichment analysis indicated that these differentially expressed genes (DEGs) were significantly associated with ATP metabolism. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis also showed that high stocking density resulted in the dysregulation of oxidative phosphorylation. Furthermore, high stocking density upregulated six lipid metabolism-related pathways. Overall, our findings, despite the limited number of samples, suggested that high stocking density led to oxidative stress and dysregulation of energy metabolism in the gills of C. quadricarinatus under rice-crayfish co-culture. Alteration in energy metabolism may be an adaptive response to adverse farming conditions.

Dietary supplementations modulate the physiological parameters, fatty acids profile and the growth of red claw crayfish (Cherax quadricarinatus).

An optimal diet is an important factor for the proper growth and health of crustaceans. However, the regulation of antioxidant activity and non-specific immunity related to the consumption of feed additives has not been studied in RC-crayfish. Triplicate groups of 20 crayfish/tank (36.72 ± 0.70 g) fed with a basal diet and sixteen experimental diets that contained five feed additives with four grade levels (40, 160, 240 and 320 mg/kg vitamin E, 2, 4, 6 and 8 g/kg nucleotides, 2, 4, 6 and 8 g/kg Haematococcus pluvialis, 5, 10, 15 and 20 g/kg arachidonic acid and 2.5, 5, 10 and 15 g/kg yeast extract) on physiological parameters, fatty acids profile and growth of Cherax quadricarinatus for a period of 70 days by using orthogonal array method (L16 45 ). The results showed that the antioxidants activity in the haemolymph and hepatopancreas were both higher in crayfish fed with diets NO. 9 to 12 than others. Also, all the diets except diets NO. 13 to 16 showed lower free radicals contents than the control group. Similarly, significantly higher non-specific immune parameters were observed in the hepatopancreas of crayfish supplementations than those fed a control diet. Biochemical parameters related to protein profile in haemolymph increased in diets NO. 9 to 12 and then decreased in control and diets NO. 13 to 16, while the highest biochemical parameters related to lipid profile except HDL-c contents in haemolymph were observed in crayfish fed the control diet. Fatty acid composition in the hepatopancreas, muscle and ovary of RC-crayfish was significantly influenced by using the combination of Vit E, NT, H. pluvialis and YP compared to the control group. Compared to all treatments, RC-crayfish fed with diets NO. 2 and 12 had significantly stimulated higher growth performance and feed utilisation. Overall, our results suggest that diets supplemented with Vit E level of 240 mg/kg, in combination with 8 g/kg NT, 4 g/kg, H. pluvialis, 5 g/kg ARA and 10 g/kg YP are the promising treatments to increase antioxidants activity, non-specific immune response, fatty acids composition and growth of RC-crayfish. However, high dietary supplementations level can reduce antioxidants activity, immunity and inhibit growth.

Activation of p53 in anoxic freshwater crayfish, Faxonius virilis.

Tumor suppressing transcription factor p53 regulates multiple pathways including DNA repair, cell survival, apoptosis and autophagy. Here, we studied the stress-induced activation of p53 in anoxic crayfish (Faxonius virilis). Relative levels of target proteins and mRNAs involved in the DNA damage response were measured in normoxic control and anoxic hepatopancreas and tail muscle. Phosphorylation levels of p53 were assessed using immunoblotting at sites known to be phosphorylated (serine 15 and 37) in response to DNA damage or reduced oxygen signaling. The capacity for DNA binding by phosphorylated p53 (p-p53) was also measured, followed by transcript analysis of a potentially pro-apoptotic downstream target, the etoposide induced (ei24) gene. Following this, both inhibitor (MDM2) and activator (p19-ARF) protein levels in response to low-oxygen stress were studied. The results showed an increase in p-p53 levels during anoxia in both hepatopancreas and tail muscle. Increased transcript levels of ei24 support the activation of p53 under anoxic stress. Cytoplasmic accumulation of Ser15 phosphorylated p53 was observed during anoxia when proteins from cytoplasmic and nuclear fractions were measured. Increased cytoplasmic concentration is known to initiate an apoptotic response, which can be assumed as a preparatory step to prevent autophagy. The results suggest that p53 might play a protective role in crayfish defense against low-oxygen stress. Understanding how anoxia-tolerant organisms are able to protect themselves against DNA damage could provide important clues towards survival under metabolic rate depression and preparation for recovery to minimize damage.

Genome-wide investigation of toll-like receptor genes (TLRs) in Procambarus clarkia and their expression pattern in response to black may disease.

As a crucial component of pattern-recognition receptors (PRRs) that recognizing pathogen-associated molecular patterns (PAMPs) and defending against invading pathogens, the Toll-like receptors (TLRs) have been paid extensive attention. While the identification and functional roles of TLRs in innate immunity have been reported in a plenty of organisms, the systematic knowledge of TLRs is still lacking in the red swamp crayfish (Procambarus clarkia). In current study, a total of 7 tlr genes were identified in P. clarkia based on the published transcriptome and genome data. The PcTLRs length varied from 939 to 1517aa and contain typical domains of TLR protein, including transmembrane region, varied LRR and TIR domains. 7 Pctlr genes were distributed in 5 chromosomes and 2 scaffolds. The expression pattern of different Pctlr genes in different tissues (hepatopancreas, gill and muscle) and in response to black may disease (BMD) showed significant difference. In addition, 5 proteins that might interact with PcTLR-2 were predicted, among them the expression pattern of dorsal and relish was consistent with Pctlr-2 in three tissues, while the other genes were not. The PcTLR-2-Dorsal/Relish pathway might play crucial roles in response to BMD infection. The results provided a theoretical foundation for further studies on the molecular mechanisms of TLRs in BMD infection in the red swamp crayfish and provided reference for the research of other crustacean species.

Impact of nitrite exposure on oxidative stress and antioxidative-related genes responses in the gills of Procambarus clarkii.

Nitrite is the major environmental pollutant in the freshwater aquaculture environment, which has a negative impact on aquatic species growth. Currently, we know that the main way nitrite enters crustaceans is through their gills. In this study, a total of 96 h acute nitrite stress (60 mg/L) experiments were conducted, and the impact of the serum biochemical parameters, gill oxidase activity and oxidative-related gene expression of red swamp crayfish were evaluated. After exposure to nitrite for 0, 6, 12, 24, 48, and 96 h, hemolymph and gills samples were taken at each time point. In the serum, acute nitrite stress significantly increased glutamic-oxaloacetic transaminase (GOT) and alanine aminotransferase (ALT) activities after 6 h of exposure, decreased total protein (TP) and albumin (ALB) levels after 24 h and 48 h of exposure, respectively. In the gills, the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were enhanced to the maximum level at 12 h, 24 h and 24 h, respectively. The contents of malondialdehyde (MDA) and lipid peroxide (LPO) were increased significantly after 12 h and 24 h exposure, respectively. In addition, the expression levels of antioxidative-related genes, including hsp70, fer and mt, were significantly upregulated in the gills after 6 h of exposure. The results indicated that acute nitrite stress changed the serum physiological status, induced oxidative stress and caused damage to gill cells in P. clarkii.

Effects of phytase supplementation of high-plant-protein diets on growth, phosphorus utilization, antioxidant, and digestion in red swamp crayfish (Procambarus clarkii).

Fish meal is increasingly being replaced by plant protein raw materials, meanwhile, it brings phytic acid, which combines with phosphorus to form phytate phosphorus and leads to a low utilization rate of phosphorus in shrimp. To solve this problem, this study investigated the effects of phytase supplementation on growth performance, phosphorus utilization, antioxidants, and digestion in red swamp crayfish (Procambarus clarkii). Crayfish (initial mean weight: 8.69 ± 0.15 g, N = 324) were randomly divided into six groups each with three replicates of 18 individuals each, and hand-fed for 8 weeks with one of six experimental diets (50 and 490 g kg-1 animal and plant protein raw material, respectively): negative control (NC; 11.0 g kg-1 phosphorus), positive control (PC; 15 g kg-1 NaH2PO4 added to NC; 14.7 g kg-1 phosphorus), and phytase supplementation diets (P1-P4: 0.1, 0.2, 0.4, and 0.6 g kg-1 phytase added to NC, respectively). The feeding trial was performed in a micro-flow water culture system. P2 showed a significantly higher weight gain rate (WGR), specific growth rate, protein efficiency ratio, and protein retention efficiency (PRE) but showed the lowest feed conversion ratio (FCR) than other groups. Broken-line regression analyses using WGR, FCR, and PRE as evaluation indices showed that the optimal dietary phytase supplementation level was 0.233, 0.244, and 0.303 g kg-1, respectively. P2 showed the highest crude protein content of whole crayfish and abdominal muscle, and phosphorus deposition rate, which was significantly higher than that in NC and PC. P3 showed the highest calcium and phosphorus contents in whole crayfish and phosphorus content in abdominal muscle, and calcium and inorganic phosphorus content in serum, which were significantly higher than those in NC. P3 showed significantly lowest serum alkaline phosphatase, alanine aminotransferase, aspartate transaminase activities, malondialdehyde content in hepatopancreas, and highest catalase activity, which were significantly lower and higher, respectively, than those in NC and PC. In summary, the addition of 0.2-0.4 g kg-1 phytase significantly improves the growth performance, feed utilization, digestive enzyme activity, and antioxidant of P. clarkii, which has a similar effect to the direct addition of NaH2PO4 at 15 g kg-1 to the feed.

Genomic structure, expression, and functional characterization of the Fem-1 gene family in the redclaw crayfish, Cherax quadricarinatus.

The Fem-1 (Feminization-1) gene, encoding an intracellular protein with conserved ankyrin repeat motifs, has been proven to play a key role in sex differentiation in Caenorhabditis elegans. In the present study, three members of the Fem-1 gene family (designating Fem-1A, Fem-1B, and Fem-1C, respectively) were cloned and characterized in the redclaw crayfish, Cherax quadricarinatus. Sequence analysis showed that all three Fem-1 genes contained the highly conserved ankyrin repeat motifs with variant repeat numbers, which shared similarity with other reported crustaceans. In addition, a phylogenetic tree revealed that the Fem-1 proteins from C. quadricarinatus were clustered with the crustacean Fem-1 homologs, and had the closest evolutionary relationship with Eriocheir sinensis. Quantitative real-time PCR (qRT-PCR) results demonstrated that Fem-1B exhibited a significant higher expression abundance in the ovary than in other tissues. In addition, a regular mRNA expression pattern of the Fem-1B gene appeared in the reproductive cycle of ovarian development. Furthermore, RNA interference experiments were employed to investigate the role of Fem-1B in ovarian development. Moreover, knockdown of Fem-1B by RNAi decreased the expression of VTG in the ovaries and hepatopancreas. In summary, this study pointed out that Fem-1B was involved in the sex differentiation process through regulating VTG expression in C. quadricarinatus, and provided new insights into the role of Fem-1B in ovary development.

Regulation of the cell cycle under anoxia stress in tail muscle and hepatopancreas of the freshwater crayfish, Orconectes virilis.

Regulation of the cell cycle is an understudied response to oxygen deprivation among crustaceans. The virile crayfish, Orconectes virilis, is a freshwater crustacean that when challenged by environmental oxygen limitation undergoes metabolic rate depression (to ~30% of normal levels) and switches to anaerobic metabolism to generate energy. To understand how crayfish regulate the cell cycle in response to anoxia, key proteins involved in cell cycle control were analyzed in muscle and hepatopancreas. At the G1/S barrier, an overall upregulation of positive regulators of cell cycle progression was indicated by the responses of G1 cyclins (cyclin D and cyclin E) and Cyclin dependent kinases (CDK4, CDK6 and CDK2) under anoxia. Although the levels of Cyclin kinase inhibitors (CKIs) at this juncture were also upregulated (P15/16 and P21 (T145) in muscle and P16 (S152) in hepatopancreas), levels of a major regulator of this phase and driver to S-phase, E2F1, were significantly higher in both tissues in conjunction with deactivation of its inhibitor, Retinoblastoma (Rb) protein. At the G2/M barrier, expression profiles of the G2 cyclin B suggested cell cycle progression despite overall trend of higher activities of checkpoint kinases, (Chk1 (S317) and Chk2 (S19)), that also negatively regulate the cyclin B-CDK1 complex via CdC25C (cell division cycle 25) whose levels remained unchanged. Overall, the present study suggests continued cell cycle progression, albeit with potential deceleration, as indicated by checkpoint kinases and kinase inhibitor profiles that might play a role in protecting tissues from apoptotic damage under chronic anoxic stress.

E2F1 Expression and Apoptosis Initiation in Crayfish and Rat Peripheral Neurons and Glial Cells after Axonal Injury.

Neurotrauma is among the main causes of human disability and mortality. The transcription factor E2F1 is one of the key proteins that determine the fate of cells. The involvement of E2F1 in the regulation of survival and death of peripheral nerve cells after axotomy has not been previously studied. We, for the first time, studied axotomy-induced changes in the expression and localization of E2F1 following axonal injury in rats and crayfish. Immunoblotting and immunofluorescence microscopy were used for the analysis of the expression and intracellular localization of E2F1 and its changes after axotomy. To evaluate whether this transcription factor promotes cell apoptosis, we examined the effect of pharmacological inhibition of E2F activity in axotomized rat models. In this work, axotomy caused increased expression of E2F1 as early as 4 h and even 1 h after axotomy of mechanoreceptor neurons and ganglia of crayfish ventral nerve cord (VNC), as well as rat dorsal root ganglia (DRG). The level of E2F1 expression increased both in the cytoplasm and the nuclei of neurons. Pharmacological inhibition of E2F demonstrated a pronounced neuroprotective activity against axotomized DRGs. E2F1 and downstream targets could be considered promising molecular targets for the development of potential neuroprotective agents.

White Spot Syndrome Virus Benefits from Endosomal Trafficking, Substantially Facilitated by a Valosin-Containing Protein, To Escape Autophagic Elimination and Propagate in the Crustacean Cherax quadricarinatus.

As the most severely lethal viral pathogen for crustaceans in both brackish water and freshwater, white spot syndrome virus (WSSV) has a mechanism of infection that remains largely unknown, which profoundly limits the control of WSSV disease. By using a hematopoietic tissue (Hpt) stem cell culture from the red claw crayfish Cherax quadricarinatus suitable for WSSV propagation in vitro, the intracellular trafficking of live WSSV, in which the acidic-pH-dependent endosomal environment was a prerequisite for WSSV fusion, was determined for the first time via live-cell imaging. When the acidic pH within the endosome was alkalized by chemicals, the intracellular WSSV virions were detained in dysfunctional endosomes, resulting in appreciable blocking of the viral infection. Furthermore, disrupted valosin-containing protein (C. quadricarinatus VCP [CqVCP]) activity resulted in considerable aggregation of endocytic WSSV virions in the disordered endosomes, which subsequently recruited autophagosomes, likely by binding to CqGABARAP via CqVCP, to eliminate the aggregated virions within the dysfunctional endosomes. Importantly, both autophagic sorting and the degradation of intracellular WSSV virions were clearly enhanced in Hpt cells with increased autophagic activity, demonstrating that autophagy played a defensive role against WSSV infection. Intriguingly, most of the endocytic WSSV virions were directed to the endosomal delivery system facilitated by CqVCP activity so that they avoided autophagy degradation and successfully delivered the viral genome into Hpt cell nuclei, which was followed by the propagation of progeny virions. These findings will benefit anti-WSSV target design against the most severe viral disease currently affecting farmed crustaceans.IMPORTANCE White spot disease is currently the most devastating viral disease in farmed crustaceans, such as shrimp and crayfish, and has resulted in a severe ecological problem for both brackish water and freshwater aquaculture areas worldwide. Efficient antiviral control of WSSV disease is still lacking due to our limited knowledge of its pathogenesis. Importantly, research on the WSSV infection mechanism is also quite meaningful for the elucidation of viral pathogenesis and virus-host coevolution, as WSSV is one of the largest animal viruses, in terms of genome size, that infects only crustaceans. Here, we found that most of the endocytic WSSV virions were directed to the endosomal delivery system, strongly facilitated by CqVCP, so that they avoided autophagic degradation and successfully delivered the viral genome into the Hpt cell nucleus for propagation. Our data point to a virus-sorting model that might also explain the escape of other enveloped DNA viruses.

Molecular characterization and functional analysis of the lysosomal cathepsin D-like gene in red swamp crayfish, Procambarus clarkii.

Aspartic proteinases are one of the four families of proteinase enzymes that are widely present in living organisms. They are involved in various physiological events, such as protein degradation, development, and host defense. However, the characterization and functional roles of aspartic proteinases remain to be elucidated in crustaceans. Here, we characterized a fragment of cathepsin D-like cDNA from red swamp crayfish, Procambarus clarkii (Pc-cathepsin D-like). The open reading frame of the Pc-cathepsin D-like gene contained 1152 bp, encoding a protein of 383 amino acid residues. We also evaluated the immunological role of the Pc-cathepsin D-like gene in vivo. Spatial distribution analysis revealed that the Pc-cathepsin D-like mRNA was high in the hepatopancreas, followed by the gut, gills, and hemocytes of P. clarkii. The expression levels of the Pc-cathepsin D-like gene increased following challenge with viral (polyinosinic: polycytidylic acid) and bacterial (lipopolysaccharides, peptidoglycan) PAMPs compared with PBS injection. The suppression of the Pc-cathepsin D-like gene by RNA interference significantly increased the expression of immune-associated genes. These results showed that the Pc-cathepsin D-like gene has an essential biological role in innate immune responses because it regulates the expression of immune-associated genes.

Linking multiple aspects of thermal performance to explore the potential for thermal resource partitioning between a native and an invasive crayfish.

Ecologists require standardized, ecologically relevant information on the thermal ecology of aquatic ectotherms to address growing concerns related to changing climates, altered habitats, and introduced species. We measured multiple thermal endpoints to investigate potential for establishment of the invasive Ringed Crayfish (Faxonius neglectus) in thermally heterogeneous habitat of the narrowly distributed endemic Coldwater Crayfish (Faxonius eupunctus). For each species, we examined the relationships between thermal endpoints at the cellular and organismal levels. We then compared results between the two species to gain insight as to the generality of linkages between cellular and organismal-level endpoints, as well as the potential for thermal niche separation between the native and potential invader. At the cellular level, we found no differences in the temperature for maximum activity of electron transport system enzymes (ETSmax) between species. At the organismal level, F. neglectus preferred significantly warmer temperatures than F. eupunctus, but this difference was small (1.3 °C) and likely to have only limited biological significance. The critical thermal maximum (CTM) did not differ between species. For both species, the thermal performance curve for ETS enzyme activity served as a useful framework to link thermal endpoints and estimate the transition from optimal to stressful temperatures - organismal thermal preference and optimal temperature estimates consistently fell below ETSmax whereas CTM estimates fell above ETSmax. Taken together, the strong similarities in thermal endpoint patterns between the two species suggest habitats thermally suitable for the native F. eupunctus will also be thermally available to expanding populations of F. neglectus, thereby increasing the opportunity for negative interactions and population effects if F. neglectus invades one of the few remaining, uninvaded, critical habitats of F. eupunctus.